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Chronic hepatitis B virus (HBV) infection may progress to cirrhosis, hepatocellular carcinoma (HCC) and end-stage liver failure with time. Interruption of this process can only be achieved through effective antiviral treatment. This approach has so far involved the use of immunomodulators such as pegylated interferon alpha (Peg-IFNα) for a finite period of up to a year and nucleos-(t)ide analogues (NUCs) for treatment over much longer periods of time. The latter act by suppressing HBV replication at the level of DNA synthesis by inhibiting the viral reverse transcriptase/DNA polymerase and causing premature termination of DNA synthesis. The ideal treatment end point is loss of HBsAg in both HBeAg+ve and HBeAg-ve patients following monotherapy. This, however, is only achievable in a minority of patients. Secondary outcomes are durable HBeAg loss and seroconversion to anti-HBe, which occur in about 18-30% of HBeAg+ve patients depending on the antiviral used, and sustained suppression of HBV-DNA accompanied by biochemical normalization and histological improvement in non-HBeAg+ve seroconverting and HBeAg-ve patients. There is therefore a need for additional direct-acting antivirals (DAAs) targeting different stages of the life cycle of the virus, as well as immunotherapeutic approaches. Such developments may pave the way for their use either alone or more likely in combination in the fight against chronic HBV infection. Such drugs or approaches, which are currently undergoing preclinical or clinical testing, are the subject of this review.

The response to interferon-alpha treatment of patients with chronic hepatitis B under the current protocol is not satisfactory. The aim of this study was to try an alternative approach to improve treatment outcome. Of 374 HBeAg-positive patients, 127 of them received 5 million units of interferon-alpha thrice weekly for 6 months and constituted the control group, while 247 in the study group received the same dosage but the duration of treatment was tailored. The study protocol provided for continuation of treatment if HBV DNA levels were continuously decreasing. The treatment ended when viral, antigenic and biochemical endpoints were reached or when HBV DNA levels were no longer decreasing. The median length of tailored treatment was 10 (range 6-24) months. The end-of-treatment response rates were 39.3% and 23.6% (P = 0.002), and after 12-month, follow-up, the sustained response rates were 40.5% and 28.3% (P = 0.013) in the study and control groups, respectively. Excluding the patients who dropped out, 228 and 115 completed a median of 40- and 44-month-long follow-up; the long-term response was thus 45.3% and 33.1% (P = 0.014) in the respective groups. Interferon-alpha treatment tailored in length demonstrated significantly increased efficacy in patients with chronic hepatitis B.

The natural history of chronic hepatitis B is dependent on the age of acquiring the hepatitis B infection. Those who are infected at adolescence or adulthood (including most of the Caucasians) tend to have stable disease after hepatitis B e antigen seroconversion with normal serum alanine aminotransaminase (ALT) and hepatitis B virus (HBV) DNA levels

Persistent infection with hepatitis G virus (HGV) or GB virus-C (GBV-C) is common and may last for years. In addition, the principal site of virus replication remains undefined. Sequencing studies of E2 in four patients showed that a hypervariable region equivalent to that of hepatitis C virus (HCV) was absent and that viral quasispecies were less frequent than in HCV infection, particularly with respect to amino acid variation. Recurrence of viraemia following interferon treatment did not result in the emergence of new quasispecies. Virus persistence therefore does not appear to be related to immune escape by strains bearing a hypervariable E2 region. We also investigated whether virus replication occurred in peripheral blood mononuclear cells. The positive-RNA strand of the virus, but no negative strand, was detected in both serum and lymphocytes. The lymphocytes harbouring the virus were CD4 and CD19 positive. Direct sequencing and cloning of amplicons from the region of the non-structural 3 (NS3) protein showed that the nucleotide sequences in lymphocytes were different from those in serum and did not represent any of the minor serum quasispecies. Although evidence of replication in lymphocytes has not been forthcoming, the differences in sequences between serum and lymphocytes suggest that circulating virus originates from a non-hepatic site, other than lymphocytes.

The aim of this study was to determine the prevalence of infection with the newly described hepatitis G virus (HGV) in a liver transplant cohort, and to establish the frequency and nature of hepatitis in those with and without HGV infection. A reverse transcriptase-polymerase chain reaction technique was employed to determine viraemia in the patients, and liver biopsies taken at different times after transplantation were assessed histologically. Hepatitis G virus RNA was detected in 47% of the liver transplant recipients investigated. Those positive for HGV had received significantly more blood or blood products than the HGV-negative patients. The frequency of abnormal liver function tests was similar in HGV-positive and HGV-negative recipients. Bile duct epithelial cell damage was more frequently seen in those with HGV viraemia. This study indicates that almost half of the liver transplant recipients in Northern England are positive for HGV, and that infection is associated with exposure to blood and blood products. It appears that, in the immunosuppressed patient, HGV does not cause clinically significant liver disease, at least up to 2 years after transplantation. If HGV infection is associated with hepatitis outside this clinical setting, it is likely that the liver damage is immunopathologically mediated rather than as a result of direct viral cytotoxicity.

This is the first double-blind controlled study of famciclovir, an oral antiviral agent, as potential therapy for chronic hepatitis B virus (HBV) carries. A fall of more than 90% in HBV DNA levels was noted in six of 11 evaluable patients treated with a 10 day course of oral famciclovir. Further studies with more prolonged therapy are ongoing.

Alpha-fetoprotein (AFP) is a marker of the presence of hepatocellular carcinoma (HCC), but is also elevated in advanced chronic hepatitis B. The detection and usage of AFP tests need to be improved. A cohort of 101 patients with advanced chronic hepatitis B and elevated AFP values was treated with entecavir (ETV) or peginterferon-alpha2a. ETV was more effective in reducing AFP levels; mean time to AFP normalization was 11.9 weeks after ETV treatment initiation vs 22.3 weeks in peginterferon treated patients (P = 0.000). An additional cohort of 93 hepatitis B virus (HBV) cirrhotic patients with elevated AFP were treated with ETV prospectively and maintained under intensive surveillance. HCC developed in 16 (17.2%) patients in whom the strongest independent predictor was a continued AFP rise in spite of ongoing treatment. In this context, nodules of sizes 10-14 mm and 15-20 mm were detected in 40% of patients each. In conclusion, HBV cirrhotic patients with rising AFP levels were at very high risk of HCC development. Early detection of minute lesions may be possible by monitoring AFP levels, whilst patients are on treatment in conjunction with enhanced computed tomography examination.

Hepatitis B virus (HBV) strains carrying the precore stop-codon mutation (A1896) have been considered among the predisposing factors for reactivation during chemotherapy for malignancies. The role of the T1762/A1764 basic core promoter (BCP) mutations has not been fully evaluated. We aimed to record any changes in HBV serological markers after reactivation, detect the presence of A1896 and BCP mutations and evaluate the type of cytotoxic drugs involved. We retrospectively screened eight patients presenting with HBV reactivation following chemotherapy for malignancies. The chemotherapy regimens used included corticosteroids (CSs), fludarabine and cyclophosphamide/adriamycine. The INNO-LiPA HBV PreCore kit was used for the detection of the A1896 and BCP mutations. Six patients who were hepatitis B surface antigen (HBsAg)-(+)/hepatitis B e antigen (HBeAg)-(-) before chemotherapy, had disease reactivation following a mean of four cycles of chemotherapy. Four survived and two died of hepatic failure. At the time of reactivation, all six patients carried the A1896 and five of them the BCP mutations. The remaining two patients were HBsAg-(-)/anti-HBs-(+)/anti-hepatitis B core (HBc)-(+)/HBeAg-(-) before chemotherapy. One of them reverted to HBeAg-(+) status but remained HBsAg-(-), while the other became HBsAg-(+)/HBeAg-(+), following three and eight cycles of fludarabine treatment, respectively. The former carried the A1896 and the latter the wild-type virus. Both died from causes associated with their haematological disease. All but one of our patients with HBV reactivation during chemotherapy carried the precore stop-codon and BCP mutations. Whether this occurs more frequently in such patients than those carrying the wild-type virus needs further investigation. Fludarabine should be added to the list of drugs inducing HBV reactivation. HBV reactivation following fludarabine treatment occurred in HBsAg-(-) patients who had been anti-HBs-(+).

Variants of hepatitis B virus (HBV), with amino acid substitutions in the major antigenic "a" determinant of hepatitis B surface antigen (HBsAg), have been described mainly in vaccinated children. In the present study in addition to vaccinated children, we have investigated Chinese blood donors positive for anti-HBc alone, and a patient with continuing liver disease after interferon-induced seroconversion to anti-HBs. Variants were detected in two of four children with break-through infections. One child had a double mutation (P142S and G145R) and the other a G145A substitution. Three of seven anti-HBc positive Chinese blood donors had a T131I substitution, whilst the interferon-treated patient had a treble amino acid substitution (P142S, G145R and N146D). The present results indicate that HBsAg variants may exist in individuals other than vaccinated children.

Summary. We describe comparative sequence analysis of 20 isolates of the recently discovered hepatitis G virus (HGV) and propose a model of the RNA secondary structure at the 3′ end of the 5′ untranslated region (UTR) of this virus. A single AUG starting at nucleotide position 552, which was in-frame and continuous with the putative polyprotein, was conserved in all 20 isolates and appeared to be the most likely site for the initiation of polyprotein synthesis. This consensus AUG was 14 amino acid residues upstream of a sequence with identity to the envelope protein El of hepatitis C virus (HCV), but the actual function of this N-terminal peptide of HGV is still not certain. None of the isolates encoded a sequence with similarity to the nucleocapsid protein of any known virus. The RNA secondary structure of the fragment under study was determined using thermodynamic modelling and validated using the results of comparative sequence analysis. Further support for the model was gained from the prediction of significant sequence and structural homology in the RNA secondary structure of the complete 5′-UTR of GB virus-A (GBV-A). A possible mechanism for translation initiation in HGV and GBV-A was suggested by the identification of features in common with the internal ribosome entry sites (IRESs) of HCV and picornaviruses.

A new member of the Flaviviridae family has recently been cloned and completely sequenced. The new virus, tentatively named hepatitis G virus (HGV) and known to be closely related to GB virus C (GBV-C), is transmitted by blood and blood products, intravenous drug use and other behaviour associated with a high risk of parenteral exposure to blood. The association of the virus with hepatitis is demonstrated by the presence of raised liver transaminase (alanine aminotransferase, ALT) levels in patients infected with HGV in the absence of other identifiable causes of hepatitis. No patient sera from groups exposed to blood and blood products were found to be positive when tested for the presence of GBV-A or GBV-B sequences, two other recently described flaviviruses. Forty-five per cent of the HGV-infected patients investigated had normal ALT suggesting the existence of a normal carrier state. Persistent infection of up to 13 years duration was observed. Co-infection with hepatitis B or hepatitis C viruses (HBV and HCV) was commonly seen presumably because of shared risk factors. None of five patients with fulminant hepatic failure was positive for HGV infection. The virus is sensitive to interferon-alpha, but sustained responses were not seen with the treatment regimens used for HBV and HCV. Viral titres increased during immunosuppression following liver transplantation and the higher levels of viraemia were in one case accompanied by elavated ALT. Whether HGV (GBV-C) replicates in the liver in some or all cases remains to be established. Preliminary data suggest that it is present within peripheral blood lymphocytes.

We tested the sera of 67 consecutive patients for hepatitis G virus (HGV) RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). These patients (42 males and 25 females, median age 35 years, range 13-64 years) had liver disease of unknown aetiology and were without markers of hepatitis (A-E) viruses or signs of genetically determined, autoimmune, alcoholic or drug-induced liver disease. The controls in this study were 110 patients (50 females and 60 males, median age 45 years, range 9-65 years) with chronic hepatitis B virus (HBV) infection (19 patients) or hepatitis C virus (HCV) infection (91 patients). Ten of 67 (14.9%) patients with cryptogenic disease were positive for HGV RNA by at least three separate tests; HGV RNA was also detected in one of 19 (5.3%) hepatitis B surface antigen (HBsAg) carriers and in nine of 91 (16.6%) patients with antibody to HCV. These data suggest that HGV occurs as frequently in HCV-infected patients as in those with cryptogenic disease. Elevated serum gamma glutamyl transpeptidase (gamma-GT) (higher than twice the normal value) and alkaline phosphatase levels were found in eight of 10 (80%) HGV RNA positive patients and in six of 57 (10.5%) HGV RNA negative patients (P < 0.0001). Five (50%) HGV RNA positive patients had non-specific inflammatory bile duct lesions. A statistically significant difference was observed between HGV RNA positive and negative patients with chronic HBV or HCV infections (P < 0.029). Therefore, the spectrum of liver disease associated with HGV is wide, but a characteristic lesion of the bile duct leading to elevation of cholestatic enzymes might be specific for this virus.

The precore stop-codon variant of hepatitis B virus (HBV) has been associated with fulminant hepatitis but is also found in patients with persistent infection and chronic hepatitis. We have examined the possibility that the severe outcome of infection in patients with fulminant disease may be a result of additional genomic variation. We sequenced the entire HBV genome from three patients of Greek and one patient of Chinese origin with fulminant hepatitis, and from two patients with hepatitis B e antigen (HBeAg) positive chronic infection from the same regions, using direct sequencing of amplified viral DNA. Three of the fulminant cases were infected with the precore stop-codon variant HBeAg negative) and the fourth with the wild-type (HBeAg) positive virus. We compared sequences from our four fulminant isolates, and an additional fulminant isolate reported by others, with HBeAg positive carriers from the same regions and 12 published HBV genomes. There was a higher number of nucleotide and amino-acid substitutions throughout the HBV genome in the precore variant fulminant sequences than in the wild type. A cluster of mutations previously identified in the X region (126-132) in sequences reported in Japanese patients and encompassing the Enhancer II-Core Promoter region (1751-1768), were not found in our patients. We conclude that although there are no changes common to all sequences of HBV isolates from fulminant cases, some of these changes are in recognized cis-acting regulatory elements, whilst others are in the immediate vicinity of such elements. The effect of these mutations on viral genome transcription must now be determined.

SUMMARY. A recombinant protein corresponding to part of the amino-terminal domain of hepatitis B virus (HBV) polymerase was expressed in Escherichia coli. Antisera raised against this protein stained hepatocytes, from human liver biopsies, predominantly in the nucleus but some cytoplasmic staining was also observed. No staining was observed in hepatocytes from uninfected patients. Liver biopsies, taken from patients who were infected with human immunodeficiency virus (HIV) as well as HBV showed more intense staining with these antisera than that seen in patients who were infected with HBV alone. The staining pattern suggests that either the whole HBV polymerase protein, or a portion encoding the amino-terminal domain, is translocated to the nucleus. This event may be an important early step in replication of the HBV genome.

Summary. The genomes of three new flavi-like viruses, GBV-A, GBV-B and GBV-C have been identified. Nucleic acid molecules corresponding to the genomes of GBV-A and GBV-B were isolated from tamarins with hepatitis which had been infected with the GB agent. RNA sequences corresponding to GBV-C have been shown to be present in sera from humans with non-A-E hepatitis. Sequence comparisons show that these three viruses are more closely related to each other and to hepatitis C virus (HCV) than to any other known viruses. Together with HCV they appear to form a discrete cluster of related viruses within the larger genus of flaviviridae. The pathological significance of these viruses and their association with hepatitis is currently emerging.